Inflammatory Diseases—Rheumatoid Arthritis
Inflammation, a reaction of the body to injury or to infectious, allergic, or chemical irritation can lead to a variety of inflammatory diseases or disorders such as inflammation associated with allergy, inflammation related to the production of nitric oxide, inflammation related to the skin, abdomen, peripheral or central nervous system, eye or tear glands, ear, nose, mouth, lung, heart, liver, pancreas, thyroid, adipose tissue, kidney, joints or blood vessels, or inflammation related to infection, trauma or autoimmunity.
Rheumatoid arthritis (RA) is a chronic, inflammatory, systemic autoimmune disease that affects about 1% of the general population in Western societies (Gabriel 2001). The disease process results in progressive destruction of joint cartilage and bone. This destruction results from immune responses and non-antigen-specific innate inflammatory processes. The disease is characterized by mono- or polyarticular joint inflammation with massive accumulation of neutrophils in the synovial fluid and tissue. The synovial neutrophils contribute to cartilage destruction by releasing proteases and generating oxidants and it is becoming more and more evident that inhibiting neutrophil infiltration into inflamed joints could be an approach to prevent progression of the disease (Hallett 2008). Current therapies for RA include non-steroid anti-inflammatory drugs (NSAIDs) for pain treatment, disease-modifying antirheumatic drugs (DMARDs) and biological agents that target specific proinflammatory cytokines, or cell surface receptors of various cell types.
There remains a need, however, for alternative pharmaceutical treatments of inflammatory diseases, especially chronic inflammatory diseases. Consequently there is a need to identify new unique targets involved in inflammatory signalling and processes, which can be used as the basis for development of new innovative therapeutic agents for the treatment, prophylaxis and prevention of inflammatory diseases.
Bile Salt-Stimulated Lipase
The bile salt-stimulated lipase (BSSL) also designated carboxyl ester lipase (CEL) or bile salt-dependent lipase (BSDL) is a lipolytic enzyme expressed in the exocrine pancreas and secreted into the intestinal lumen in all species so far investigated. In some species, including the human, BSSL is also expressed by the lactating mammary gland and secreted with the milk. BSSL has broad substrate specificity with capacity to hydrolyze a variety of different substrates, e.g. cholesteryl esters, tri-, di-, and monoacylglycerols, fat-soluble vitamin esters, phospholipids, galactolipids and ceramides (Hui and Howles 2002). The physiological function of BSSL was originally thought to be confined to the small intestine and hydrolysis of dietary fat (Hernell et al. 1997). The high abundance of BSSL in pancreatic juice (up to 5% of total protein content) and the ability of BSSL to hydrolyze a broad spectrum of lipids have led researchers to suggest a variety of functions for BSSL in lipid digestion and absorption. BSSL has a key role in the absorption of cholesteryl esters (Fält et al. 2002), verified in mice lacking the BSSL (CEL gene) (Howles et al. 1996). While this is considered its main function in the human adult it is likely to contribute also to triglyceride digestion and absorption in the newborn infant (Lindquist and Hernell 2010).
BSSL was found to be present in low, but significant levels in serum of healthy individuals (Bläckberg et al. 1985) and current research has implicated that BSSL is involved in lipoprotein metabolism and modulation of atherosclerosis (Hui and Howles 2002). The potential function, or even the question if elevated levels of circulating BSSL is a risk factor for, or protects against atherosclerosis is not clear. A surprisingly strong positive association between BSSL, assayed as cholesterol esterase activity, and total—as well as low-density lipoprotein (LDL)-cholesterol levels in serum was first reported (Hui and Howles 2002). BSSL was then shown to be associated with smooth muscle cells (SMCs) within atherosclerotic plaques and to induce vascular SMC proliferation in vitro (Auge et al. 2003). A study, using transgenic mice, demonstrated that macrophage expression of BSSL is pro-atherogenic, favouring cholesteryl ester accumulation and foam cell formation (Kodvawala et al. 2005). Judged by these studies BSSL would be a risk factor for atherosclerosis. On the other hand, BSSL reduces lysophosphatidylcholine content in oxidized LDL, thereby reducing accumulation of oxidized LDL in macrophages (Hui and Howles 2002), and it has been suggested to play a physiological role in hepatic selective uptake and metabolism of high density lipoprotein cholesteryl esters by direct and indirect interactions with the scavenger receptor BI pathway (Camarota et al. 2004), which implicates that BSSL in serum protects against atherosclerosis.
The BSSL Protein
The human BSSL protein (encoded by the CEL gene) is a single-chain glycoprotein of 722 amino acids (Nilsson et al. 1990). The enzyme is synthesised as a precursor of 742 amino acids with a signal peptide of 20 amino acids. Two bile salt-binding sites regulating the activity of the enzyme and the resistance to proteases have been postulated (Hui 1996) as well as a sphingolipid binding domain (SBD) (Aubert-Jousset et al. 2004).
Schematically the enzyme can be divided into two parts:
i) The N-terminal domain with a striking homology to acetylcholinesterase and some other esterases. In this part the proposed catalytic triad (Ser194 (included in the motif GESAG), Asp320 and His435) are found as well as a N-glycosylation site, Asn187, a heparin-binding site (postulated to be located at position 1-100) and the two intra chain disulfide bridges (Cys64-Cys80 and Cys 246-Cys257). The heparin binding ability has been found to be located in the part of the molecule consisting of amino acids 1-445 (Spilburg et al. 1995) and the heparin binding domain may, in fact, be a three-dimensional structure composed of different sequences. The heparin binding properties of BSSL is thought to be important for interactions with cell membranes, exemplified by intestinal cell membranes (Fält 2002).
ii) The C-terminal part (encoded by exon 11) with a variable number of tandem repeats (VNTR)-region containing similar but not identical repeats (11 amino acids). The most common human form contains 16, but there is a variation in number of repeats both between individuals and alleles (Lindquist et al. 2002). The repeats are followed by an extra tail of 11 amino acids (this tail is longer in the corresponding rat and mouse enzyme). The repeats are proline-rich and the presence of aspartic acid in every repeating unit and glutamic acid in some, render this region highly acidic and contributes to the low iso-electric point of the protein. The number of proline-rich repeats has been reported to vary extensively between species, typically ranging from three in mouse and the cow, four in the rat to 16 in humans and 39 in the gorilla (Hui and Howles 2002; Madeyski et al. 1999). This diversity in number of repeated units can explain the observed size differences of the protein between species; the mouse BSSL is a 74 kDa protein while the human BSSL, which is extensively glycosylated across the repeated region, has an apparent molecular mass of 120-140 kDa; the repeats carry most of the 15-35% carbohydrate of the protein. The varying apparent molecular mass can be explained both by the number of repeats and differences in glycosylation (Lindquist et al. 2002). It has been shown by analysing the isolated C-terminal part of human milk BSSL (amino acids 528-722) that probably only 10 out of 16 repeats in human milk BSSL are O-glycosylated (Wang et al. 1995).
It has been suggested that the repeats may have a functional role in protecting BSSL from proteolytic degradation and that their O-glycosylation is important for secretion of the enzyme (Bruneau et al. 1997). The oligosaccharides in the C-terminal region contain Lewis x and Lewis b and less Lewis a antigenic structures. Owing to those blood-group-related antigenic determinants, the C-terminal region of BSSL may have an adhesive function in cell-cell interactions, as illustrated by its antimicrobial effects (Naarding et al. 2006; Ruvoën-Clouet et al. 2006). On the other hand, the repeated region may be less important for catalytic activity, activation by bile salts and heparin binding (Hui 1996).
The C-tail has also been suggested to be an important structural part by binding to a lectin-like receptor (LOX-1) on the surface of intestinal endothelium cells (Fayard et al. 2003). The heparin binding site(s) forms the other binding part, and these binding sites have a pivotal role in the mechanism of action for BSSL in different cellular environments and cell stages.
Vascular BSSL
Comparison of BSSL VNTR genotype and serum lipid phenotype revealed an association between the number of repeats and serum cholesterol profile (Bengtsson-Ellmark et al. 2004). While it is possible that the repeat polymorphism is merely a genetic marker for lipid profile, it is also possible that it has functional role in determining plasma lipid composition.
A wider role for BSSL in lipid metabolism is implicated by the presence of BSSL in human plasma and aortic tissue. The source of circulating BSSL has been discussed extensively. Human macrophages and endothelial cells were shown to synthesize and secrete the enzyme (Hui and Howles 2002). Conversely, in another study BSSL within atherosclerotic lesions was associated with smooth muscle cells (SMCs) but not with activated macrophages or endothelial cells (Augé et al. 2003). In yet another study, BSSL injected into rat intestinal loops was advocated to be internalized by enterocytes, transferred through the cells and released into the circulation (Bruneau et al. 2003). Based on these data it was proposed that circulating BSSL originates from the pancreas. However, it has been further shown that neither does the BSSL serum level increase after a meal of breast milk, nor does it differ between breastfed and formula fed human infants, although in the newborn breast milk is the major source of BSSL, while it is absent from infant formula (Bläckberg et al. 1985; Shamir et al. 2003).
An association of BSSL with apolipoprotein B-containing lipoproteins in human plasma has been reported (Bruneau et al. 2003), which together with the observation that BSSL is present in the human aorta and has the ability to modify low density lipoprotein (LDL) and high density lipoprotein (HDL) composition and reduce the atherogenicity of oxidized LDL (oxLDL) by decreasing their lysophosphatidylcholine (lysoPC) content (Shamir et al. 1996), invoked a potential new role for BSSL as a protective factor in the development of atherosclerosis. LysoPC is a major phospholipid component in oxLDL and is generated by oxidation and fragmentation of polyunsaturated fatty acids esterified to the sn-2 position of the PC molecule, followed by hydrolysis of the shortened fatty acyl residue by LDL-associated phosolipase A2 (PLA2) and BSSL. Although lysoPC constitutes only 1-5% of total PC in non-oxLDL, oxidative modification of LDL can raise this proportion to as high as 40-50%. LysoPC acts as a chemoattractant for monocytes, induces monocyte adhesion to the vascular endothelium and promotes macrophage proliferation, which eventually leads to foam cell formation. Due to its effects on lysoPC, it has been suggested that BSSL may interact with cholesterol and oxidized lipoproteins to modulate the progression of atherosclerosis (Hui and Howles 2002).
However, the fact that BSSL is found and accumulated in atherosclerotic lesions, and the fact that monocytes as well as macrophages (or SMC having a macrophage phenotype) express and secrete BSSL, indicate that these cells may be a possible source of the accumulated BSSL. The mechanism behind a pathophysiological role of BSSL in macrophages is suggested to be the function of BSSL as a ceramidase (Hui and Howles 2002) by its reduction of ceramide and lysophosphatidylcholine levels leading to increased cholesteryl ester accumulation in response to atherogenic lipoproteins resulting in increased atherosclerosis lesion size in vivo. This is in line with the study by Kodvawala et al. (2005), who by using in vivo models showed that BSSL expression in macrophages promotes cholesteryl ester synthesis and accumulation in response to modified LDL and increases atherosclerosis lesions in apoE deficient mice.
The Response to Retention Hypothesis of Atherosclerosis
Many of the processes implicated in the early stages of atherogenesis including endothelial damage, lipoprotein oxidation and macrophage and VSMC (vascular smooth muscle cells) proliferation are individually not sufficient to lead to lesion development. The response-to-retention hypothesis suggests that subendothelial retention of atherogenic lipoproteins is the trigger for all of these processes which are in fact normal physiological responses to the accumulation of lipids.
While the major determinant of initial retention of LDL is likely to be the proteoglycan composition within the subendothelial space, BSSL may facilitate and enforce retention once the lesion has started to form by acting as a molecular bridge between the subendothelial proteoglycans and lipoproteins (WO 2005/095986). The BSSL that is bound to the components of the extracellular matrix can act as bridging molecules in the retention of LDL, as suggested for Lipoprotein lipase (LPL) (Pentikainen et al. 2002).
BSSL in Platelets
Recently BSSL was found to be stored in blood platelets and released upon platelet activation (Panicot-Dubois et al. 2007). Moreover, BSSL was shown to induce calcium mobilization in platelets and to enhance thrombin-mediated platelet aggregation and spreading.
In a mouse thrombosis model (laser-induced injury), BSSL accumulated in arterial thrombi in vivo—at sites of vessel wall injury. When CXC chemokine receptor 4 (CXCR4) was antagonized, the accumulation of BSSL was inhibited and thrombus size was reduced. In BSSL knockout mice (BSSL-KO) tail bleeding times were increased in comparison with those of wild-type mice. These data suggest that BSSL modulates thrombus formation by interacting with CXCR4 on platelets.
CXCR4 belongs to the G-protein-coupled receptor (GPCR) gene family, and upon activation CXCR4 induces downstream signalling by several different pathways; e.g. CXCR4 binding of the chemokine ligand SDF-1 activates G-protein mediated signalling and induces cellular chemotactic responses (Clemetson et al. 2000). CXCR4 is also known to interact with HIV-1 and to act as a co-receptor for entry of the virus into cells. The binding of HIV-1 to CXCR4 is mediated via a domain denoted the V3 loop present on HIV-1 gp120. The BSSL protein contains a region that is structurally related to the V3-loop of gp120. This region, called the V3-like loop domain (amino acids 361-393) (Aubert-Jousset et al. 2004) was proposed to mediate the binding of BSSL to CXCR4 on platelets.
In summary, there are both confusing and conflicting result regarding the source and function of BSSL in plasma and aortic tissue.
EP 1840573 reports on differences in gene expression pattern between NOD (non-obese diabetic) mice positive or negative for insulin autoantibodies. 125 differentially expressed genes were identified, one of them being the CEL gene encoding BSSL. The differentially expressed genes are identified as having utility in early diagnosis of a pre-inflammatory state of autoimmune diseases, such as type I diabetes.
The differentially expressed genes are further suggested to be targets for the treatment of autoimmune diseases having a pre-inflammatory phase. It is well known in the art that expression of numerous genes is altered as a consequence of the development of a specific disease, as demonstrated in EP 1840573. However, all such differentially expressed genes can not be considered to be the cause of the development of the disease. On the contrary the identification of the causative gene(s), if at all existing, requires further complicated investigations. EP 1840573, even if identifying BSSL as potential marker for inflammatory disease, fails to identify BSSL as a cause for the development of inflammatory disease.